滋阴清热复方、NAC与mitoQ对地塞米松所致MRL/lpr小鼠肾脏线粒体功能异常影响的研究

Effects of Compound Ziyin Qingre Prescription, NAC and mitoQ on abnormal renal mitochondrial function in dexamethasone-induced MRL/lpr mice

目的探讨滋阴清热复方、N-乙酰半胱氨酸(N-Acetyl-L-cysteine,NAC)与米托蒽醌甲磺酸盐(mitoquinone mesylate,mitoQ)对地塞米松所致MRL/lpr小鼠线粒体功能异常影响。方法50只8周龄的MRL/lpr小鼠随机分为狼疮鼠组、地塞米松组、滋阴清热复方+地塞米松组、NAC+地塞米松组、mitoQ+地塞米松组,每组10只;另选Balb/c小鼠为空白对照组,共10只;分别于干预12周后,取肾脏组织,通过实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,qRT-PCR)与蛋白质印迹(Western-blot,WB)检测线粒体基因细胞色素C氧化酶亚基1(MT-CO1),线粒体自噬基因PTEN诱导激酶1(PTEN-induced putative kinase 1,PINK1)、白细胞介素(interleukin,IL)-1β的mRNA及蛋白表达水平;比色法检测丙二醛(malondialdehyde,MDA)、超氧歧化酶(superoxide dismutase,SOD)表达差异。结果与正常鼠组相比,狼疮鼠组肾脏线粒体呼吸链基因MT-CO1表达降低[qRT-PCR:(1.00+0.12)比(0.39±0.05);WB:(1.73±0.14)比(0.75±0.08),t值分别为9.61,10.60,P值均<0.05]、线粒体复合物Ⅳ活性减弱[(9.37±0.32)比(7.12±0.26),t=9.53,P<0.05];SOD活性降低[(20.62±3.97)比(15.04±0.33),t=2.80,P<0.05],MDA含量升高[(21.90±2.39)比(41.51±4.74),t=7.40,P<0.05];线粒体自噬基因PINK1表达升高[qRT-PCR:(0.99±0.17)比(1.42±0.05);WB:(0.80±0.06)比(1.37±0.07),t值分别为4.11,10.57,P值均<0.05],炎症因子IL-1β表达升高[qRT-PCR:(1.04±0.32)比(3.43±0.66);WB:(2.48±0.15)比(3.20±0.24),t值分别为6.48,4.45,P值均<0.05],差异具有统计学意义。与狼疮鼠组相比,地塞米松组肾脏线粒体呼吸链基因MT-CO1表达[qRT-PCR:(0.39±0.05)比(0.19±0.05);WB:(0.75±0.08)比(0.36±0.08),t值分别为5.49,5.79,P值均<0.05]、线粒体复合物Ⅳ活性减弱[(7.12±0.26)比(5.83±0.39),t=5.56,P<0.05],SOD(U/mg)活性降低[(15.04±0.33)比(10.28±1.16),t=7.94,P<0.05]、MDA(μmol/mg)蛋白含量升高[(41.51±4.74)比(65.91±6.22),t=6.24,P<0.05];线粒体自噬基因PINK1表达升高[qRT-PCR:(1.42±0.05)比(1.87±0.04);WB:(1.37±0.07)比(1.74±0.14),t值分别11.95,5.79,P值均<0.05],炎症因子IL-1β表达降低[qRT-PCR:(3.43±0.66)比(1.27±0.37),P<0.05;WB:(3.20±0.24)比(1.31±0.31),t值分别为5.69,8.36,P值均<0.05],差异具有统计学意义。与地塞米松组比,滋阴清热复方+地塞米松组、NAC+地塞米松组与mitoQ+地塞米松组表达肾脏线粒体呼吸链基因MT-CO1表达升高[qRT-PCR:(0.19±0.05)比(1.09±0.06)、(0.88±0.05)、(1.51±0.15),t值分别为22.45,19.16,16.93,P值均<0.05;WB:(0.36±0.08)比(1.28±0.05)、(1.09±0.06)、(1.67±0.10),t值分别为16.59,12.41,16.82,P值均<0.05],线粒体复合物Ⅳ活性升高[(5.83±0.39)比(9.05±0.64)、(8.58±0.10)、(10.80±0.67),t值分别为7.83,9.63,11.73,P值均<0.01],SOD活性升高[(10.28±1.16)比(22.98±0.61)、(21.47±0.88)、(37.48±1.35),t值分别为19.45,15.45,30.67,P值均<0.05]、MDA含量降低[(65.91±6.22)比(21.63±4.86)、(21.30±1.85)、(22.51±4.36),t值分别为11.22,11.78,11.43,P值均<0.05];线粒体自噬基因PINK1表达降低[qRT-PCR:(1.87±0.04)比(1.04±0.09)、(1.41±0.30)、(0.41±0.08),t值分别为16.40,29.86,2.93,P值均<0.05;WB:(1.74±0.14)比(1.03±0.10)、(0.83±0.11)、(0.99±0.05),t值分别为7.17,8.90,8.74,P值均<0.05],差异具有统计学意义,炎症因子IL-1β表达差异无统计学意义[qRT-PCR:(1.27±0.37)比(1.67±0.15)、(0.84±0.11)、(1.61±0.26),t值分别为2.04,2.20,1.52,P均>0.05;WB:(1.31±0.31)比(1.25±0.10)、(1.84±0.30)、(1.11±0.18),t值分别为0.31,2.15,0.98,P值均>0.05]。与NAC+地塞米松组相比,滋阴清热复方+地塞米松组肾脏线粒体呼吸链基因表达MT-CO1表达升高,差异具有统计学意义[qRT-PCR:(0.88±0.05)比(1.09±0.06);WB:(1.09±0.06)比(1.28±0.05),t值分别为5.31,4.44,P值均<0.05];与mitoQ+地塞米松组相比,滋阴清热复方+地塞米松组线粒体基因MTCO1表达下降[qRT-PCR:(1.51±0.15)比(1.09±0.06),WB:(1.67±0.10)比(1.28±0.05),t值分别为5.23,5.91,P值均<0.05],SOD活性下降[(37.48±1.35)比(22.98±0.61),t=19.61,P<0.05],差异具有统计学意义。结论滋阴清热复方可以减轻糖皮质激素所致的线粒体功能异常,改善细胞和器官功能,其机理可能通过抗氧化实现。

Objective Exploring the effects of Compound Ziyin Qingre Prescription,N-Acetyl-L-cysteine(NAC)and Mitoquinone mesylate(mitoQ)on abnormal mitochondrial function in dexamethasone-induced MRL/lpr mice.Methods Fifty 8-week-old MRL/lpr mice were randomly divided into lupus mouse group,dexamethasone group,Compound Ziyin Qingre Prescription+dexamethasone group,NAC+dexamethasone group,and mitoQ+dexamethasone group.Ten mice in each group;Balb/c mice were selected as blank control group,10 mice in total,after 12 weeks of intervention respectively,the kidney tissues were taken and the mRNA and protein expression levels of mitochondrial gene MT-CO1,mitochondrial autophagy gene PINK1,inflammatory factor interleukin(IL)-1βwere detected;colorimetric method was used to detect the difference of malondialdehyde(MDA)and superoxide dismutase(SOD)expression.Results Compared with normal mice,the renal mitochondrial respiratory chain gene MT-CO1 expression[RT-qPCR:(1.00+0.12)vs(0.39±0.05);WB:(1.73±0.14)vs(0.75±0.08),t values were 9.61,10.60,both P values<0.05],mitochondrial complexⅣactivity and SOD activity were decreased[(9.37±0.32)vs(7.12±0.26),(20.62±3.97)vs(15.04±0.33),t values were 9.53,2.80,both P values<0.05],MDA was increased[(21.90±2.39)vs(41.51±4.74),t=7.40,P<0.05],mitochondrial autophagy gene PINK1 expression was increased[RT-qPCR:(0.99±0.17)vs(1.42±0.05);WB:(0.80±0.06)vs(1.37±0.07),t values were 4.11,10.57,both P values<0.05]and inflammatory factor IL-1βexpression was increased[RT-qPCR:(1.04±0.32)vs(3.43±0.66),WB:(2.48±0.15)vs(3.20±0.24),t values were 6.48,4.45,both P values<0.05]in the lupus group.Compared with MRL/Lpr mice,the dexamethasone group showed decreased mitochondrial respiratory chain gene expression MT-CO1[RT-qPCR:(0.39±0.05)vs(0.19±0.05),WB:(0.75±0.08)vs(0.36±0.08),t values were 5.49,5.79,both P values<0.05],mitochondrial complexⅣ[(7.12±0.26)vs(5.83±0.39),t=5.56,P<0.05]activity and SOD activity[(15.04±0.33)vs(10.28±1.16),t=7.94,P<0.05],increased MDA[(41.51±4.74)vs(65.91±6.22),t=6.24,P<0.05];increased mitochondrial autophagy gene PINK1 expression[RT-qPCR(1.42±0.05)vs(1.87±0.04),t=11.95,P<0.05;WB:(1.37±0.07)vs(1.74±0.14),t=5.79,P<0.05],and decreased inflammatory factor IL-1βexpression[RT-qPCR:(3.43±0.66)vs(1.27±0.37),t=5.69,P<0.05;WB:(3.20±0.24)vs(1.31±0.31),t=8.36,P<0.05]in kidney.Compared with the dexamethasone group,the Compound Ziyin Qingre Prescription+dexamethasone group,NAC+dexamethasone group and mitoQ+dexamethasone group expressed increased renal mitochondrial respiratory chain gene expression MT-CO1[RT-qPCR:(0.19±0.05)vs(1.09±0.06),(0.88±0.05),(1.51±0.15),t values were 22.45,19.16,16.93,all P values<0.05;WB:(0.36±0.08)vs(1.28±0.05),(1.09±0.06),(1.67±0.10),t values were 16.59,12.41,16.82,all P values<0.05],mitochondrial complexⅣactivity[(5.83±0.39)vs(9.05±0.64),(8.58±0.10),(10.80±0.67),t values were 7.83,9.63,11.73,all P values<0.05]and SOD(U/mg)activity[(10.28±1.16)vs(22.98±0.61),(21.47±0.88),(37.48±1.35),t values were 19.45,15.45,30.67,all P values<0.05],decreased MDA(μmol/mg)[(65.91±6.22)vs(21.63±4.86),(21.30±1.85),(22.51±4.36),t values were 11.22,11.78,11.43,all P values<0.05];decreased mitochondrial autophagy gene PINK1 expression[qPCR:(1.87±0.04)vs(1.04±0.09),(1.41±0.30),(0.41±0.08),t values were 16.40,29.86,2.93,all P values<0.05;WB:(1.74±0.14)vs(1.03±0.10),(0.83±0.11),(0.99±0.05),t values were 7.17,8.90,8.74,all P values<0.05],and no difference in inflammatory factor IL-1βexpression[qPCR:(1.27±0.37)vs(1.67±0.15),(0.84±0.11),(1.61±0.26),t values were 2.04,2.20,1.52,all P values>0.05;WB:(1.31±0.31)vs(1.25±0.10),(1.84±0.30),(1.11±0.18),t values were 0.31,2.15,0.98,all P values>0.05].Compared with the NAC+dexamethasone group,the renal mitochondrial respiratory chain gene expression MT-CO1 was increased in the Compound Ziyin Qingre Prescription+dexamethasone group[RT-qPCR:(0.88±0.05)vs(1.09±0.06);WB:(1.09±0.06)vs(1.28±0.05),t values were 5.31,4.44,P<0.05];compared with the mitoQ+dexamethasone group,the mitochondrial gene expression MTCO1[RT-qPCR:(1.51±0.15)vs(1.09±0.06);WB:(1.67±0.10)vs(1.28±0.05),t values were 5.23,5.91,both P values<0.05]was decreased and SOD activity[(37.48±1.35)vs(22.98±0.61),t=19.61,P<0.05]was decreased in the Compound Ziyin Qingre Prescription+dexamethasone group.Conclusion Compound Ziyin Qingre Prescription can alleviate GC-induced abnormal mitochondrial function and improve cell and organ function,the mechanism of which may be achieved through antioxidation.